Cancer presents a major public health issue concerning the significant number of affected individuals and the relatively high mortality rates, including for many diseases of origin. Breast and prostate cancers rank among the cancers with the highest incidence and morbidity (Weir et al, 2003). Both cancers are initially hormone-dependent with prostate cancer cells activated by androgen, which binds the androgen receptor (AR), and breast cancer cells activated by estrogen, which binds the Estrogen receptor (ER). Both forms of cancer respond to anti-hormone therapy. In time, though, hormone-independent cancers develop and remain a serious challenge to therapy. Clearly, a key underlying mechanism of breast and prostate cancer, including the switch of breast and prostate cancer to hormone independence, is improper control of transcription. This results in increased levels of c-myc in both prostate and breast cancers (Chrzan et al., 2001; Ellwood-Yen et al., 2003). Numerous other genes are also upregulated or downregulated (Gelmann and Semmes, 2004).
Considering the key function of transcription, it is no surprise that several best-selling drugs approved in the United States target transcription regulatory circuits (Emery et al., 2001). However, methods for directed targeting of core transcription components by therapeutic agents have yet to be established. The challenge thereof is in the complexity of the machinery, and in purification of large amounts of high-quality RNA mammalian Polymerase II (RNAP). To date RNAP, the eukaryotic motor of transcription involved in generating all mRNA, has been produced with low yields and in a relatively impure state (Thompson et al., 1990). However, assay development involving RNAP and its interacting factors require high yields and highly pure proteins.
Mammalian RNAP comprises 12 polypeptides with a mass of over 500 kD. Regulation of this complex can occur during initiation, elongation of the RNA transcript, or termination of the process. In addition, gene expression is dependent upon the structure of DNA, such as the positioning of nucleosomes, which is a basic structural unit comprised of DNA and histones (Belotserkovskaya et al., 2004; Khorasanizadeh, 2004). DNA-binding transcription factors are also involved in effectuating an increase or decrease in gene expression.
One such DNA-binding transcription factor over-expressed in breast and prostate cancers is the c-myc oncogene (Jenkins et al., 1997). Myc is a major player in prostate cancer, as evidenced by transgenic mice expressing human c-Myc in the mouse prostate. All transgenic mice developed murine prostatic intraepithelial neoplasia followed by invasive adenocarcinoma (Ellwood-Yen et al., 2003). Indeed, growth of human androgen-independent prostate cancer (AIPC) cells, androgen recepter (AR)-positive or -negative, requires c-myc expression (Bernard et al., 2003). In breast cancer c-myc plays an equally important role and is also amplified and overexpressed (Blancato et al., 2004).
C-myc may have its effect by altering downstream cellular processes. For example, it was suggested that up-regulation of telomerase activity observed in prostate tumors might be conferred through stimulation of telomerase catalytic sub-unit expression by c-myc. (Latil et al., 2000). It was also shown that the involvement of myc may be directly on the androgen receptor gene, as a Myc consensus site in the AR coding region is involved in androgen-mediated up-regulation of AR messenger RNA (Grad et al, 1999).
Since over-expression of c-myc exists in uncontrolled breast and prostate cancer proliferation, gene therapy strategies that employ expression of c-myc antisense have been attempted. In one such case, a single direct injection of MMTV-antisense c-myc viral media into established DU145 tumors in nude mice resulted in a 94.5% reduction in tumor size compared to controls, with two animals having complete regression. The mechanism for the anti-tumor effects was suppression of c-myc mRNA resulting in down-regulation of the bcl-2 protein. (Steiner et al., 1998). Other efforts include the use of antisense phosphorodiamidate morpholino oligomers directed against c-myc in a PC-3 androgen-independent human prostate cancer xenograft murine model and testing for safety in a Phase I human clinical study. A 75-80% reduction in tumor size was observed in treated animals compared with control groups. Safety trials in humans showed no toxicity or serious adverse events, and phase II trials are underway (Iversen et al., 2003).
TFIIS: A Putative Transcription Factor Target Upstream of c-myc
C-myc is overexpressed in breast and prostate cancer and is targeted for therapy. Interestingly, regulation of c-myc expression can occur during elongation of the growing RNA strand (Keene et al., 1999). During transcription of the Myc gene, RNAP must traverse polyT stretches in the DNA, which are known to cause arresting of transcription. This arresting is regulated by the transcription factor TFIIS, which directly binds RNAP allowing for read through of the PolyT stretch. Arresting of c-myc during transcription elongation has been shown, and poly T sequences that are TFIIS responsive in c-myc are the key intrinsic factor in arresting (Keene et al., 1999; Bains et al., 1997; Kerppola and Kane, 1988; Kerppola and Kane, 1990; Astrom et al., 1999). In addition, TFIIS is considered a candidate gene for human malignancies involving deletions in the chromosomal region, 3p21.3→3p22 TFIIS (Astrom et al., 1999). TFIIS is therefore a good target for cancer drug development, including breast and prostate cancer drug development.
Another overexpressed breast cancer-related protein is c-fos. c-fos is critical for MCF7 breast cancer cell growth and is an effective target for a breast cancer vaccine (Lu et al., 2005; Luo et al., 2003). c-fos is reported to contain a poly T arrest site similar to that of c-myc (Plet et al., 1995). TFIIS allows for readthrough of poly T tracts and, as a result, its inhibition may reduce c-fos expression.
Targeting Transcription and Potential Toxicology of Non-cancerous Cells
A fair question concerning toxicological effects of inhibiting higher order transcription factors is in place. By inhibiting transcription factors, both normal and cancer cells may be affected. Insight from clinical trials involving the inhibition of higher order transcription factors sheds light on this issue.
One such case involves the HDAC (histone deacetylase) inhibitors. As mentioned above, nucleosome positioning is a key part of chromosomal structure, and acetylation or de-acetylation of the histone component of nucleosomes affects transcription. Inhibitors to HDACs cause cell cycle arrest, differentiation and/or apoptosis of many tumors, which may be useful for chemotherapy (Yoshida et al., 2003), and they are in clinical trials (Leone et al., 2003). Apparently, HDAC inhibitors have shown impressive antitumor activity in vivo with remarkably little toxicity in preclinical studies (Vigushin and Coombes, 2002). Other chromatin-remodeling transcription factors may therefore also be valid targets for cancer therapy, such as the SWI/SNF chromatin remodeling complex (Reyes et al., 1997). As such, a global regulator is shown to present clinically with little toxicity despite affecting transcription of numerous genes.
A second case is Flavopiridol, a kinase and higher order transcription inhibitor (Kelly et al., 2002). One of its targets, pTEFb, like TFIIS, is a transcription elongation factor that directly interacts and phosphorlyates RNAP. Flavopirodol inhibits transcript elongation, effectively eliminating transcription, yet it is well tolerated in clinical trials as an anti-cancer agent (Aklilu et al., 2003). Flavopiridol, though, inhibits several cell cycle kinases, and a search for drugs that specifically affect transcription elongation alone is in need. Interestingly, even siRNA inhibition of RNAP itself is tolerated in animal models (Fluiter et al., 2003).
One reason why transcription inhibition may be tolerated is that normal cells can maintain homeostasis after transcription factor inhibition for some period of time, whereas cancer cells may require the state of aberrant transcription to survive. Disrupted transcriptional homeostasis or reduced amounts of oncogenic proteins may cause either differentiation or apoptosis as it has in the case of a conditional Myc model (Jain et al., 2002). In addition, inhibiting specific transcription factors may shut down only a subset of genes. For example, yeast cells with TFIIS deletions are viable (Nakanishi et al., 1995).
WO 98/41648 concerns methods of inhibiting growth of a cell by administering inhibitors active on an allele of a gene that is vital for cell growth and viability, and the reference provides a list of potential genes, including some transcription factors.
WO 01/72777 relates to methods for treating disease associated with decreased or increased expression of a transcription factor by administering an antagonist or agonist to a particular polypeptide, including transcription factors.
WO 02/22660 is directed to methods to treat individuals by administering an antibody to particular gene products, including transcription factors.
The present invention satisfies a need to provide novel targets in the challenging field of cancer therapeutics.